Bacteria cells present an outstanding ability to rapidly react to various changes in their growth environment. The number of useful antibiotic agents is decreasing fast. Thus, there is an urgency for finding alternative ways to deal with the crisis.
The natural environment of bacteria is often characterized by changes in nutrient availability. When bacterial cells are deprived of an amino acid or carbon source, changes in many cellular processes occur. Immediately after sensing the inception of amino acid starvation, bacteria respond pleiotropically with the stringent response, which was initially described for Escherichia coli in 1961. The first observed feature of the stringent response was the intracellular accumulation of two unusual phosphorylated derivatives of GTP and GDP (collectively named (p) ppGpp), within a few seconds after amino acid starvation (Cashel and Gallant, 1969; Cashel et al., 1969, Cashel et al. 1970). Other features of the stringent response include inhibition of rRNA and tRNA synthesis, inhibition of replication initiation and cell division, suppression of the active transport of many metabolites, transcriptional upregulation of genes encoding enzymes involved in amino acid biosynthesis (Cashel, 1996), and induction of the rpoS gene, which encodes the stationary phase sigma factor (Gentry et al., 1993).
In E. coli, the mutation causing the relaxed phenotype, which fails to accumulate (p) ppGpp during amino acid starvation, was mapped to the relA gene which encodes an 84 kDa protein, RelA (Metzger et al., 1988). The RelA protein is a ribosome-associated (p) ppGpp synthetase that is activated in response to amino acid starvation. Synthesis of (p) ppGpp has been characterized as a pyrophosphoryl group transfer of the β and γ phosphates from an ATP donor to the ribose 3′ hydroxyl of GTP (or GDP) (Cashel, 1996). For this reaction to occur in vitro, purified RelA requires mRNA, functional ribosomes paused during elongation at a ‘hungry codon’, and uncharged cognate tRNA bound at the acceptor site of that hungry codon (Cashel, 1996). In cell extracts, RelA is found associated to only a small fraction of the ribosomes (about 1%) (Pedersen and Kjeldgaard, 1977). RelA is thus a ribosome-dependent enzyme that senses environmental amino acid levels by monitoring the amount of uncharged tRNA present in the cell, and accordingly synthesizes the intracellular second-messenger, (p) ppGpp (Haseltine, 1973; Metzger et al., 1988).
In addition to RelA, a second gene product, SpoT, is involved in (p) ppGpp metabolism in E. coli. SpoT is a cytosolic protein that functions as a (p) ppGpp synthetase upon carbon or fatty acid limitation (Gentry and Cashel, 1995; Metzger et al., 1989; Seyfzadeh et al., 1993). Equally important, SpoT also acts as a ribosome-independent (p) ppGpp hydrolase that degrades the (p) ppGpp back to GDP(GTP) and pyrophosphate, thus catalyzing a reaction opposing the synthesis of (p) ppGpp from GDP(GTP) and ATP (Metzger et al., 1989). Residual (p) ppGpp synthesis found in a ΔrelA mutant (relA1) is abolished in a ΔrelAΔspoT (“double null”) mutant (Xiao et al., 1991). Cells with this double deletion show a complex phenotype, such as loss of ability to grow on amino acid-free minimal medium, morphological alterations and more (Xiao et al., 1991).
It has been found that in several Gram-positive bacteria, only one relA/spoT paralog exists which is capable of carrying out both the (p) ppGpp-synthetase and (p) ppGpp-hydrolase functions (Mechold et al., 1996; Mechold and Malke, 1997; Mittenhuber, 2001; Wendrich and Marahiel, 1997). Deletion of this one gene in gram-positive bacteria creates a phenotype resembling that of the ‘double null’ E. coli cells (Wendrich and Marahiel, 1997). This bifunctional gene product was even found to be essential in the highly pathogenic Staphyloccous aureus (Gentry et al., 2000). The gram-negative Myxococcus xanthus has both relA and spoT analogs, which appear to be involved in fruiting-body development and spore formation in response to starvation (Harris et al., 1998). Rel/Spo genes are absent in Archaea, in agreement with the transcriptional system being closer to that of eukaryotes, but they are again found in the genome of plants, e.g. Arabidopsis thaliana, where they play a role in activating a (p) ppGpp-mediated stress response (van der Biezen et al., 2000; Givens et al., 2004; Takahashi et al., 2004).
The crystal structure of the N-terminal domain (NTD) of Rel/Spo from Streptococcus equisimilis (Relseq) reveals two enzyme conformations. This domain has two sub-domains, each with a catalytic site, one responsible for the synthesis of (p) ppGpp, the other for its hydrolysis. The X-ray structure of the NTD also revealed the binding sites for two guanosine nucleotides.
RelA was found to be involved in the virulence, biofilm formation and survival of many bacteria species. Because RelA and its homologues are completely absent in mammals, new antibacterial compounds could be designed based on the known X-ray structure of the NTD of Relseq.
At the onset of sporulation, B. subtilis replicates its chromosome and remodels the daughter chromosomes into an elongated axial filament structure, which results in polar septum formation that divides the developing cell into two distinct cells with very different fates, forespore and mother cell. The forespore ultimately becomes the spore, whereas the mother cell nurtures the developing spore until it is discarded by lysis once morphogenesis is complete (Errington et al.). CodY, a highly conserved protein in gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP.
The stringent response appears to participate in inactivation of CodY in two indirect ways. First, synthesis of (p) ppGpp is at the expense of GTP. Second, Freese et al. showed that (p) ppGpp inhibits IMP dehydrogenase, the first enzyme of the GMP synthesis pathway. Both effects of the stringent response cause a reduction in the GTP pool. Inhibition of ppGpp synthesis may inhibit the drop in GTP level, and consequently CodY would not be inactivated, leading to the inhibition of sporulation.
The applicants of the present invention have previous reported in the use of ppGpp analogues that inhibit the synthetase activity of Rel proteins from both Gram positive and Gram negative bacteria (Wexselblatt et al., WO 2009/116044). The notion is to avoid ppGpp synthesis by inhibiting Rel proteins from both Gram positive and Gram negative bacteria. ppGpp is responsible for penicillin tolerance (Rodionov et al.), and plays an important role in the stringent response as described above. It is hypothesized that by inhibiting this mechanism bacteria will be less tolerant and more susceptible to antibiotic treatment.
There is an ongoing and unmet need in the art to identify new compounds acting as anti-bacterial agents. In addition, there is a need to combat the growing problem of bacterial resistance to anti-bacterial agents.